Structural characterization and evaluation of antimicrobial and cytotoxic activity of six plant phenolic acids

Phenolic acids still gain significant attention due to their potential antimicrobial and cytotoxic properties. In this study, we have investigated the antimicrobial of six phenolic acids, namely chlorogenic, caffeic, p-coumaric, rosmarinic, gallic and tannic acids in the concentration range 0.5–500 μM, against Escherichia coli and Lactobacillus rhamnosus. The antimicrobial activity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Additionally, the cytotoxic effects of these phenolic acids on two cancer cell lines, the colorectal adenocarcinoma Caco-2 cell line and Dukes’ type C colorectal adenocarcinoma DLD-1 cell line was examined. To further understand the molecular properties of these phenolic acids, quantum chemical calculations were performed using the Gaussian 09W program. Parameters such as ionization potential, electron affinity, electronegativity, chemical hardness, chemical softness, dipole moment, and electrophilicity index were obtained. The lipophilicity properties represented by logP parameter was also discussed. This study provides a comprehensive evaluation of the antimicrobial and cytotoxic activity of six phenolic acids, compounds deliberately selected due to their chemical structure. They are derivatives of benzoic or cinnamic acids with the increasing number of hydroxyl groups in the aromatic ring. The integration of experimental and computational methodologies provides a knowledge of the molecular characteristics of bioactive compounds and partial explanation of the relationship between the molecular structure and biological properties. This knowledge aids in guiding the development of bioactive components for use in dietary supplements, functional foods and pharmaceutical drugs.


Introduction
There has been a growing interest in natural compounds as potential pharmaceutics or chemical agents including antioxidant or preservatives that can be applied in food industry.One of Wareth et al. p-CA showed no antimicrobial action against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Aspergillus niger or Candida albicans but exhibited ability to inhibit growth of Bacillus cereus (IZ = 9 mm) [23].p-CA antibacterial mechanism was investigated by Lou et al.The results showed that p-CA caused an efflux of potassium ions from cells, indicating an increase in membrane permeability.It was revealed that p-CA caused membrane damage, leading to the release of cellular contents.Furthermore, their studies showed that p-CA can bind to DNA, potentially affecting its structure and function leading to death of a cell [24].p-CA exhibits significant cytotoxic abilities against various cancer cell lines (S2 Table ).p-CA demonstrated its great efficacy in inhibiting the growth of HeLa cells, with an IC 50 value of 1 μg/mL.HT-29 cells exhibited an IC 50 value of 25 μg/mL suggesting a substantial cytotoxic effect.Karakurt et al. evaluated cytotoxic effects of p-CA using the Alamar Blue assay.The study determined that p-CA exhibited cytotoxic abilities against DLD-1 cells with an IC 50 value of 254.3 μg/mL Caco-2 cells with IC 50 of 714.6 μg/mL [25].
Gallic acid (S2 Fig) (GA) is the most popular of trihydroxybenzoic acids that is found in the leaves of pomegranate bark, gall nuts, Indian gooseberry, witch hazel, sumac, tea leaves, oak bark, and many other plants, both in free form and as part of tannin molecules.It occurs in abundance in processed beverages such as red wine or green tea [33].GA exhibits significant antimicrobial abilities against a wide range of microorganisms, including bacteria and fungi (S2 Table ).In studies conducted by Kalinowska et al it displayed a MIC of 3 mg/mL against E. coli, B. subtilis, Salmonella enteritidis, and C. albicans, however S. aureus was more resistant to GA antibacterial abilities with MIC 6 mg/mL [34].Furthermore, it generates an IZ of 11 mm against Pseudomonas fluorescens, 13.2 mm against P30-4, 15 mm against Pseudomonas fragi, and 14.8 against Pseudomonas putida [35].In case of fungal infections, GA demonstrated its effectiveness by inhibiting the growth of Streptococcus mutans and Pseudomonas aeruginosa, with MIC values of 62.5 μg/mL and 100 μg/mL, respectively [36].One of GA antimicrobial mechanisms is the inhibition of efflux pumps, which contribute to antimicrobial resistance in bacteria [37].GA might cause impairing in the permeability of cellular membranes and also altering its charge or hydrophobicity.This might lead to DNA degradation, impairment of gene expression and cell signaling, disruption of energy metabolism, and ATP depletion [38,39].GA has been evaluated for its cytotoxicity in various cell lines using different assays (S2 Table ).In the research conducted by Pham et al. on A2780, H460, A431, MCF-7, and MCF10A cell lines using the MTT assay, the concentration of 50 μM GA did not exhibit any cytotoxic properties after 72 h of incubation [40].However, studies by Rezaei-Seresht et al. on breast cancer MCF-7 line showed that GA possess cytotoxic activity against this specific cell line with IC 50 value of 18 μg/mL [18].GA has been found to increase the expression of p53 and disrupts mitochondrial membranes, which may lead to cytochrome C release, caspase-3 activation, and subsequent apoptotic cell death.Additionally, GA can mediate in DNA damage-related regulations and overexpression of the P21 gene.The P21 can promote cellular differentiation and inhibition of cell proliferation [18].
Tannic acid (TA) (S2 Fig) is a naturally occurring plant polyphenol that can be found in various plants, notably in certain tree species such as sumac, oak, and myrobalan, particularly their barks, are rich sources of it.Additionally, tannic acid can be found in galls formed on plants [41].It has a rich history of medicinal use, including the treatment of diarrhea, burns, and rectal disorders [42].Additionally, it has been associated with various health benefits, including modulating immune responses, reducing allergen levels, and accelerating blood clotting [42][43][44].Tannic acid exhibits antibacterial abilities against various bacterial strains.The minimum inhibitory concentrations [MICs] of tannic acid in studies conducted by Sahiner et al. were as follows: 60 μg/mL for P. aeruginosa, 60 μg/mL for B. subtilis, 40 μg/mL for S. aureus and 70 μg/mL for C. albicans.However, in the same study TA expressed no antimicrobial activity against E. coli [44].On the other hand, tannic acid demonstrated inhibitory effects against E. coli with an average diameter of 13.33 mm and against S. aureus with a diameter of 10.16 mm [45].Moreover, TA exhibits a moderately inhibitory effect against S. typhimurium 400 μg/mL (MQIC value, minimal quorum inhibitory concentration).In case of methicillinresistant Staphylococcus aureus, tannic acid showed a MIC of 64 μg/mL [46].TA penetrates bacterial cell walls, disturbing bacterial metabolism, and potentially causing DNA leakage.Moreover, TA limits bacterial growth by hindering sugar and amino acids absorption [47].TA demonstrates cytotoxic abilities against various cancer cell lines (S2 Table ).TA induces cytotoxicity by caspase-mediated apoptosis, suppresses fatty acid synthase and inhibits epidermal growth factor receptor pathway.It reduces viability, metastatic potential and stemness of cancer cells [48][49][50].
In this study, we will evaluate the antimicrobial activity of these six natural phenolic acids against a bacterial strains living in the digestive system, i.e.Escherichia coli and Lactobacillus rhamnosus based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay.Studies of antimicrobial activity aim to determine the suitability of the tested compounds as potential preservatives with antimicrobial properties against selected food pathogens (E.coli).Moreover, the studies on the effect of the analysed compounds on L. rhamnosus were conducted, which are part of the human intestinal microflora to exclude any negative effects on beneficial probiotic bacteria.We will also investigate those phenolic acids cytotoxic effects of on two colorectal adenocarcinoma cell lines: Caco-2 and DLD-1 cell lines in order to analyze potential anticancer activity of studied compounds.Additionally, some structural parameters of the phenolic acids such as lipophilicity coefficient (logP) and reactivity of molecules on the basis of parameters calculated in Gaussian 09W program such aslike ionization potential, electron affinity, electronegativity, chemical hardness, chemical softness, dipole moment and electrophilicity index.The six natural phenolic compounds were chosen on purpose.They have proven biological properties such as antioxidant, antimicrobial or chemopreventive evaluated in different model systems of bacteria or cell lines (S1 and S2 Tables).The chosen ligands are derivatives of two important aromatic acids, i.e. benzoic and cinnamic acids which differ in the presence of vinyl moiety between the aromatic ring and carboxylic group.Moreover they differ in the number of hydroxyl substituents in the ring (i.e.p-coumaric, caffeic and gallic acid), some of them are caffeic acid esters (i.e.rosmarinic and chlorogenic acids) or gallic acid ester (i.e.tannic acid).All this structural difference influence on the solubility, lipophilicity and reactivity of molecules.Therefore the following questions may arise: (1) does the number of -OH substituents in the aromatic ring is the main factor that affect the antimicrobial and cytotoxic activity of compounds (toward selected bacteria and cell lines), (2) does the conjugation of caffeic and gallic acid with other molecules influence on the physicochemical and biological properties of molecules, (c) do the selected experimental (logP) and theoretical (e.g.energy of HOMO or LUMO orbitals) correlates with the biological activity of molecules?

Quantum-chemical calculations
The density functional theory (DFT) calculations were performed in the gas phase with approximation of the isolated molecules using the Gaussian 09 [51] program running on PC computer and Gauss View [52] molecular visualization program.To calculate the geometrical structure of the compounds the B3LYP method with 6-311++G(d,p) basis set was used.The exception was represented by a molecule of tannic acid, where the HF631/3-21G method was employed.The optimized structures were possessed the minimum in the energy with no imaginary infrared wavenumbers.

Bacterial strains
Escherichia coli (ATCC 25922) and Lactobacillus rhamnosus (ATCC 53103) were obtained from the American Type Culture Collection (Manassas, VA, USA).E. coli (Gram-negative bacteria) and L. rhamnosus (Gram-positive bacteria) were grown overnight in Mueller Hinton II Broth at 37˚C.The next day, the overnight cultures were diluted in fresh MH II Broth to obtain 10 8 CFU/mL (CFU-colony forming units).For the antimicrobial activity was used the inoculum where the suspension of E. coli and L. rhamnosus cells was 10 6 CFU/mL.
Cytotoxicity assay.The cytotoxicity assay was performed according to the method of Carmichael et al using MTT reagent [54].Activity of 5-CQA, CA, p-CA, GA, TA and RA in the concentration range from 0.5μM to 500μM, was studied in colorectal adenocarcinoma Caco-2 cell line and Dukes' type C colorectal adenocarcinoma DLD-1 cell line obtained from ATCC.5-CQA, CA, p-CA, RA, GA and TA were stored at temperature 4˚C and the stock solution was prepared by dissolving it in TrisHCl buffer prior analysis.GloMax1-Multi Microplate Multimode Reader was used in order to measure the absorbance (570 nm).The viability of DLD-1 cells and Caco-2 cells was calculated as a percentage of control non-treated cells [54].

Statistical analysis
All data are given as me an values ±SD (standard deviation).Differences between treatments and untreated control human cells were analyzed by one-way ANOVA, followed by Dunnett's procedure for multiple comparisons.Significant effects are represented by p � 0.05 (*), p � 0.01 (**), p � 0.001 (***).

The theoretical electronic parameters and lipophilicity
The calculated dipole moment, the energy of the HOMO and LUMO orbitals and the electronic parameters (such as ionization potential (I), electron affinity (A), electronegativity (χ), chemical hardness (η), softness (S), chemical potential (μ) and electrophilicity (ω) indexes calculated on the basis of the HOMO and LUMO orbital's energy) [55] were obtained for the studied compounds (Fig 1).The parameters are used to predicting the electronic charge transfer within the molecule, reactivity, and stability of the compounds (S3 Table and Table 1) [55].Because for tannic acid the electronic parameters were obtained at different theoretical level than for the other ligands, they can't be directly compared within the whole series of the compounds.
The HOMO-LUMO gap values increase in the following order: RA < 5-CQA < CA < p-CA < GA, indicating the decrease in the bioreactivity of studied molecules in above series (Table 1).As a result, it can be inferred that among the studied compounds, rosmarinic acid is the most chemically active, while gallic acid is the least active.The ionization potential decreases and the value of the energy of HOMO orbital increases in same the series: p-CA !GA !5-CQA !CA !RA what means the rise in the electron donating properties in the above series of ligands in the redox reactions.
The presented MEP surface (Fig 2 ) describes the overall molecular charge distribution and allows to predict the sites for electrophilic and nucleophilic attack.The red region in the MEP plot shows an electron-rich site with electrophilic reactivity that covers the oxygen atoms in carbonyl group and phenolic OH groups.Whereas the blue color indicates an electron-deficient region which is the site of the nucleophilic attack and it is mainly located in the region of the hydrogen atom of the phenolic OH groups.

Lipophilicity
The logarithmic coefficient of substance distribution (log P) in the water-n-octanol system is a crucial parameter used to assess the hydrophilicity/lipophilicity of chemicals.Factors like bioaccumulation and toxicity are largely correlated with logP.Regarding most of the analyzed phenolic acids, the collected experimental data and calculated log P values are consistent with each other (Table 2).Based on the experimental logP values, the compounds can be arranged in order of increasing lipophilicity as follows: 5-CQA< GA< CA< RA< p-CA< TA.GA and 5-CQA logP experimental values of 0.70 and 0.30 respectively indicates their low lipophilicity (Table 2).LogP experimental values for RA, CA and p-CA are in the range of 1-2 this means that these compounds possess moderately lipophilic properties.The experimental logP value of 4.84 for TA indicates it's high lipophilic properties.pKa is a measure of the acidity or basicity of a compound.In the context of lipophilicity, pKa can provide information about the ionization state of a compound at different pH values.Compounds with lower pKa values are more likely to be ionized at physiological pH (around 7.4), while compounds with higher pKa values are more likely to be in their neutral, non-ionized form.Therefore, based on experimental data and calculated values, it can be deduced that the molecular and monodeprotonated forms of GA, RA, 5-CQA, p-CA and CF are predominant within the range of physiological pH.Among those phenolic compounds 5-CQA and RA possessed lowest pKa experimental value (3.42 and 3.57 respectively), this is due to high number of the hydroxyl groups they possess.The presence of functional groups that can act as hydrogen bond donors or acceptors enhances the hydrophilicity of a compound.Compounds containing hydroxyl and carboxylic groups have the ability to form hydrogen bonds with water molecules in an aqueous environment, influencing their solubility in water.However tannic acid possesses multiple hydroxyl groups and exhibits a pKa value of 8.5.As a result, tannic acid is significantly less acidic and highly lipophilic compared to the other studied phenolic compounds.This heightened hydrophobicity and low acidity can primarily be attributed to its strong propensity to form hydrogen bonds with itself.  .In the case of E. coli, it was observed that CA, p-CA and 5-CQA caused an insignificant increase of the bacteria growth with rising concentration of tested acids and the highest value did not exceed 16% (Fig 3).For GA the average relative viability of E. coli was approximately of 1 to 10% compared to untreated cells.After the application of TA at the concentrations from 62.5 to 500 μM one can observe a relative decrease in the cell viability approximately of 1 to 10% compared to the control cells.In the studied range of concentration the selected phenolic acid showed no effect on E. coli [44].

Antimicrobial activity
On the other hand, the studied phenolic compounds had differentiated effects on L. rhamnosus viability.In the studied range of concentration 0.98-500 μM GA, 5-CQA, CA, RA significantly stimulated the tested bacteria strain (only in case of RA at the highest concentration 500 μM no stimulation was observed).Especially for CA, 5-CQA and RA a significant increase of L. rhamnosus viability with increasing concentration of the tested acids was observed and the highest value for the bacteria did not exceed 180%.These phenolic compounds possessed the lowest logP among the tested compounds which increase in the order GA!5-C-QA!CA!RA (0.70-1.60).The values of the energy of the HOMO orbitals (EHOMO) calculated for these compounds increase in the same order as well.In turn, after the application of TA on L. rhamnosus at the lowest concentrations from 0.98 to 15.63 μM, an increase in relative cell viability of 40 to 60% was observed, while at TA concentrations from 31.25 to 500 μM there was a decrease in the relative cell viability of 20 to 80% compared to the control untreated cells.For p-CA the inhibitory effect on L. rhamnosus viability was observed only in the case of  the concentration 500 μM.The observation correlates with the value of logP and EHOMO which are respectively the highest and which is the lowest for these two phenolic acids (comparing the rest of studied phenolic acids), i.e. for TA: logP = 4.86, -EHOMO = -8,554 eV and p-CA: logP = 1.69;EHOMO = -6.431eV.The obtained results suggest that the phenolic compounds in the studied concentration range did not significantly influence the viability of E. coli, which indicates that these compounds do not stimulate the growth of a potentially pathogenic bacterium naturally present in the human microbiome.On the other hand GA, CA, 5-CQA, p-CA, RA possess prebiotic properties toward L. rhamnosus in the concentration range 0.98-250 μM.For TA this stimulatory effect was observed in the concentration range 0.98-15.63μM.

Cytotoxic activity
As it was shown in Figs 4 and 5, studied compounds did not exert significant cytotoxic effect on Caco-2 cells.5-CQA caused increases in relative Caco-2 cell viability even in 200μM concentration.After 48h treatment decreases in studied parameter were observed in lower concentrations (from 0.5μM to 10μM).In case of p-CA no significant decreases in cell viability as compared to control untreated cells were noticed.Relatively more significant effect was observed in case of p-CA after 48h treatment, when decreases below control level were observed in all analyzed concentrations.It should be underline that studied compounds were more effective against DLD-1 cells that towards Caco-2 cells (Figs 4 and 5).Statistically significant decreases in relative cell viability were observed under the influence of p-CA already in 0.5μM concentration.After 48h incubation 26% decline in cells viability as compared to control untreated cells was noticed.DLD-1 cells treated with RA showed decrease in cell viability already in 5μM concentration.
As shown in Figs 4 and 5, in Caco-2 cell line cytotoxic effect was observed rather after 48h incubation with both studied compounds.In DLD-1 cell line incubation with especially TA in all analyzed concentrations leads to statistically significant decreases in cell viability.However, 5μM of GA in Caco-2 cell line caused an increase in relative cell viability.Similar effect was observed in case of TA treatment after 48h incubation time in the lowest tested concentration.TA in 200 μM concentration caused almost 50% decline in Caco-2 cell viability after 48h incubation.More significant inhibitory effect of studied compounds was observed in DLD-1 cell line than in Caco-2 cell line.Decrease by about 50% was noticed in DLD-1 cells in the concentration of 200μM for both compounds.It should be also mentioned that in case of DLD-1 cell line we did not observe any increases as compared to control untreated cells.From the group of studied compounds TA, GA and RA were especially active inDLD-1 cell line.Obtained results are in accordance with literature data indicating anticancer properties of phenolic compounds [12].Differences between the individual tested compounds may result from their chemical structure and thus their ability to penetrate cell membranes.The research results  presented in the literature, similarly to our results, indicate differences between the tested cell lines, which are partly due to differences in the genetic and metabolic profile of cancer cells [12,18,19].The main difference between the Caco-2 and DLD-1 lines is their origin.Caco-2 cells represent Dukes' stage B, which means that the cancer has grown into the muscular layer of the intestine, while DLD-1 cells represent Dukes' stage C, which means that the cancer has metastasized to at least one lymph node in the intestinal area.By considering gene expression in both lines, differences between both lines were identified, which translate into their response to pro-apoptotic factors [66].

Conclusions
In the study it was observed that the tested compounds did not affect the viability of E.coli in the tested concentration range (0.98-500 μM), which means that they do not stimulate the growth of a potentially pathogenic bacterium naturally present in human microbiome.Additionally, GA, CA, 5-CQA, p-CA, RA possessed prebiotic properties toward L. rhamnosus in the concentration range 0.98-500 μM.For TA the prebiotic effect was observed at the lower concentration range 0.98-15.63μM.The lipophilicity (described by the logP parameter) of the phenolic compounds was correlated with their antibacterial activity toward L. rhamnosus.The compounds (i.e.TA, p-CA) with the highest values of logP exhibited an inhibitory effect on microbial growth at lower concentration levels than the phenolic compounds with the higher logP.What is interesting the calculated energy of HOMO orbital reflected changes in the lipophilicity of these compounds and may be used as a additional parameter to describe the biological activity of phenolic compounds toward L. rhamnosus.The cytotoxic activity of the phenolic acids was tested in the same concentration range as the antimicrobial.Based on the obtained results, it can be concluded that a significantly better response to the applied treatments was obtained in the case of the DLD-1 line than Caco-2, and the most effective in relation to both cell lines was TA.On the other hand, CA showed the lowest biological activity, especially in the Caco-2 line.The highest cytotoxic properties of TA toward DLD-1 cell line correspond with the highest lipophilicity and stability of the molecule compared to other tested phenolic compounds.Although these two parameters are important factors determining the biological activity of chemicals, the explanation of the high activity of TA is more complicated and is related to other mechanisms involved in induction of cell apoptosis like generation of reactive oxygen species, regulation of apoptotic and anti-apoptotic proteins, suppression and promotion of oncogenes or inhibition of matrix metalloproteinases.The aspect of possible induction of apoptosis is one of the most important elements of modern therapy from the point of view of eliminating cancer cells.The vast majority of the tested compounds seem to be promising potential anticancer agents, but they require further analysis at the molecular level to understand the mechanisms of their action.

Fig 3
Fig 3  shows the influence of p-CA, CA, GA, 5-CQA, RA and TA on the concentration range 0.98-500 μM on tested E. coli and L. rhamnosus strains.The obtained results are in agreement with S1 Table.In the case of E. coli, it was observed that CA, p-CA and 5-CQA caused an insignificant increase of the bacteria growth with rising concentration of tested acids and the highest value did not exceed 16% (Fig3).For GA the average relative viability of E. coli was approximately of 1 to 10% compared to untreated cells.After the application of TA at the concentrations from 62.5 to 500 μM one can observe a relative decrease in the cell viability approximately of 1 to 10% compared to the control cells.In the studied range of concentration the selected phenolic acid showed no effect on E. coli[44].On the other hand, the studied phenolic compounds had differentiated effects on L. rhamnosus viability.In the studied range of concentration 0.98-500 μM GA, 5-CQA, CA, RA significantly stimulated the tested bacteria strain (only in case of RA at the highest concentration 500 μM no stimulation was observed).Especially for CA, 5-CQA and RA a significant increase of L. rhamnosus viability with increasing concentration of the tested acids was observed and the highest value for the bacteria did not exceed 180%.These phenolic compounds possessed the lowest logP among the tested compounds which increase in the order GA!5-C-QA!CA!RA (0.70-1.60).The values of the energy of the HOMO orbitals (EHOMO) calculated for these compounds increase in the same order as well.In turn, after the application of TA on L. rhamnosus at the lowest concentrations from 0.98 to 15.63 μM, an increase in relative cell viability of 40 to 60% was observed, while at TA concentrations from 31.25 to 500 μM there was a decrease in the relative cell viability of 20 to 80% compared to the control untreated cells.For p-CA the inhibitory effect on L. rhamnosus viability was observed only in the case of